MscS inactivation and recovery are slow voltage-dependent processes sensitive to interactions with lipids.

Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension the channel population readily opens, but under prolonged action of moderate tension it inactivates. The inactivated state is non-conductive and tension insensitive, which suggests that the gate becomes uncoupled from the lipid-facing domains. Because the distinct opening and inactivation transitions are both driven from the closed state by tension transmitted through the lipid bilayer, here we explore how mutations of two conserved positively charged lipid anchors, R46 and R74, affect 1) the rates of opening and inactivation and 2) the voltage dependences of these transitions. Previously estimated kinetic rates for opening-closing transitions in wild-type MscS at low voltages were 3-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that MscS activation exhibits a shallow nearly symmetric dependence on voltage, whereas inactivation is substantially augmented and recovery is slowed down by depolarization. Conversely, hyperpolarization impedes inactivation and speeds up recovery. Mutations of R46 and R74 anchoring the lipid-facing helices to the inner interface to an aromatic residue (W) do not substantially change the activation energy and closing rates, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data clearly delineate the activation-closing and the inactivation-recovery pathways and strongly suggest that only the latter involves extensive rearrangements of the protein-lipid boundary associated with the uncoupling of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery suggests that membrane potential is one of the factors that regulates osmolyte release valves by putting them either on "ready" or "standby" based on the cell's metabolic state.

On the lipid dependence of bacterial mechanosensitive channel gating in situ.

For bacterial mechanosensitive channels acting as turgor-adjusting osmolyte release valves, membrane tension is the primary stimulus driving opening transitions. Because tension is transmitted through the surrounding lipid bilayer, it is possible that the presence or absence of different lipid species may influence the function of these channels. In this work, we characterize the lipid dependence of chromosome-encoded MscS and MscL in E. coli strains with genetically altered lipid composition. We use two previously generated strains that lack one or two major lipid species (PE, PG, or CL) and engineer a third strain that is highly enriched in CL due to the presence of hyperactive cardiolipin synthase ClsA. We characterize the functional behavior of these channels using patch-clamp and quantify the relative tension midpoints, closing rates, inactivation depth, and the rate of recovery back to the closed state. We also measure the osmotic survival of lipid-deficient strains, which characterizes the functional consequences of lipid-mediated channel function at the cell level. We find that the opening and closing behavior of MscS and MscL tolerate the absence of specific lipid species remarkably well. The lack of cardiolipin (CL), however, reduces the active MscS population relative to MscL and decreases the closing rate, slightly increasing the propensity of MscS toward inactivation and slowing the recovery process. The data points to the robustness of the osmolyte release system and the importance of cardiolipin for the adaptive behavior of MscS.

The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel MscS.

Vibrio cholerae adapts to osmotic down-shifts by releasing metabolites through two mechanosensitive (MS) channels, low-threshold MscS and high-threshold MscL. To investigate each channel's contribution to the osmotic response, we generated ΔmscS, ΔmscL, and double ΔmscL ΔmscS mutants in V. cholerae O395. We characterized their tension-dependent activation in patch-clamp, and the millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models describing osmolyte and water fluxes. We illustrate the sequence of events and define the parameters that characterize discrete phases of the osmotic response. Survival is correlated to the extent of cell swelling, the rate of osmolyte release, and the completeness of post-shock membrane resealing. Not only do the two channels interact functionally, but there is also an up-regulation of MscS in the ΔmscL strain, suggesting transcriptional crosstalk. The data reveal the role of MscS in the termination of the osmotic permeability response in V. cholerae.

Polymer-extracted structure of the mechanosensitive channel MscS reveals the role of protein-lipid interactions in the gating cycle.

Membrane protein structure determination is not only technically challenging but is further complicated by the removal or displacement of lipids, which can result in non-native conformations or a strong preference for certain states at the exclusion of others. This is especially applicable to mechanosensitive channels (MSC's) that evolved to gate in response to subtle changes in membrane tension transmitted through the lipid bilayer. E. coli MscS, a model bacterial system, is an ancestral member of the large family of MSCs found across all phyla of walled organisms. As a tension sensor, MscS is very sensitive and highly adaptive; it readily opens under super-threshold tension and closes under no tension, but under lower tensions, it slowly inactivates and can only recover when tension is released. However, existing cryo-EM structures do not explain the entire functional gating cycle of open, closed, and inactivated states. A central question in the field has been the assignment of the frequently observed non-conductive conformation to either a closed or inactivated state. Here, we present a 3 Å MscS structure in native nanodiscs obtained with Glyco-DIBMA polymer extraction, eliminating the lipid removal step that is common to all previous structures. Besides the protein in the non-conductive conformation, we observe well-resolved densities of four endogenous phospholipid molecules intercalating between the lipid-facing and pore-lining helices in preferred orientations. Mutations of positively charged residues coordinating these lipids inhibit MscS inactivation, whereas removal of a negative charge near the lipid-filled crevice increases inactivation. The functional data allows us to assign this class of structures to the inactivated state. This structure reveals preserved lipids in their native locations, and the functional effects of their destabilization illustrate a novel inactivation mechanism based on an uncoupling of the peripheral tension-sensing helices from the gate.